This invention relates generally to the field of virology and cancer therapy. This disclosure provides vectors in which an encoding region for glycosyltransferase is linked to a genetic element that controls transcription in a tumor or tissue specific fashion.
Many forms of cancer are intractable to traditional courses of radiation or small molecule pharmaceuticals. Considerable interest has evolved in developing gene therapy vectors as chemotherapeutic agents.
A broad variety of therapeutic genes are currently under investigation in preclinical and in clinical studies (Walther et al., Mol. Biotechnol. 13:21, 1999). The candidate genes have very different origins and different mechanisms of actionxe2x80x94which include cytokine genes, genes coding for immunostimulatory molecules/antigens, genes encoding bacterial or viral prodrug-activating enzymes (suicide genes), and tumor suppressor genes.
Some of the putative vectors are based on adenovirus. U.S. Pat. Nos. 5,631,236 and 6,096,718 (Baylor College of Medicine) cover a method of causing regression in a solid tumor, using a vector containing an HSV thymidine kinase (tk) gene, followed by administration of a prodrug such as ganciclovir. U.S. Pat. No. 6,096,718 (Baylor College of Medicine) relates to the use of a replication incompetent adenoviral vector, comprising an HSV tk gene under control of the xcex1-lactalbumin promoter.
U.S. Pat. Nos. 5,801,029 and 5,846,945 (Onyx Pharmaceuticals) relate to adenovirus in which the E1a gene has been altered so as not to bind and inactivate tumor suppressor p53 or RB. This prevents the virus from inactivating tumor suppression in normal cells, which means the virus cannot replicate. However, the virus will replicate in cells that have shut off p53 or RB expression through oncogenic transformation.
U.S. Pat. No. 5,998,205 (GTI/Novartis) pertains to a tissue-specific replication-conditional adenovirus, comprising a transcriptional regulatory sequence (such as the xcex1-fetoprotein promoter) operably linked to adenovirus early replication gene. U.S. Pat. No. 5,698,443 (Calydon) provides replication-conditional adenoviruses controlled by the PSA promoter. Alemany et al. (Cancer Gene Ther. 6:21, 1999) outline complementary adenoviral vectors for oncolysis. One vector contains cis replication elements and E1a under control of a tissue-specific promoter. The supplemental vector contains all other trans-acting adenovirus replication genes. Coinfection leads to controlled killing of hepatocarcinoma cells.
International Patent Publication WO 98/14593 (Geron) describes an adenovirus construct in which the tk gene is placed under control of the promoter for telomerase reverse transcriptase (TERT). This gene is expressed at high levels in cancer cells of any tissue type, and the vector renders cancer cell lines susceptible to toxic effects of ganciclovir. WO 00/46355 (Geron) describes an oncolytic virus having a genome in which a TERT promoter is linked to a genetic element essential for replication or assembly of the virus, wherein replication of the virus in a cancer cell leads to lysis of the cancer cell.
Koga et al. (Hu. Gene Ther. 11:1397, 2000) propose a telomerase-specific gene therapy using the hTERT gene promoter linked to the apoptosis gene Caspase-8 (FLICE). Gu et al. (Cancer Res. 60:5359, 2000) reported a binary adenoviral system that induced Bax expression via the hTERT promoter. They found that it elicited tumor-specific apoptosis in vitro and suppressed tumor growth in nude mice.
Other vectors are based on herpes family viruses, such as herpes simplex type 1 and 2. U.S. Pat. No. 5,728,379 (Georgetown University) relates to replication competent HSV containing a transcriptional regulatory sequence operatively linked to an essential HSV gene. Exemplary is the IPC4 gene under control of the pro-opiomelanocortin promoter.
Other vectors are based on the retrovirus family. U.S. Pat. No. 5,997,859 and EP 702084 B1 (Chiron) pertain to replication-defective recombinant retrovirus, carrying a vector construct capable of preventing, inhibiting, stabilizing or reversing infections, cancer, or autoimmune disease. The virus directs expression of an enzyme not normally expressed in the cells that converts a compound into a cytotoxic form. Exemplary is the HSV tk gene. WO 99/08692 proposes the use of reovirus in treating cancer, particularly ras-mediated neoplasms.
These proposed therapeutic agents are not currently approved for commercial use in the United States. There is a need to develop new constructs to improve efficacy and specificity of cancer treatment.
This invention provides a system for killing cancer cells in vitro or in vivo, using a polynucleotide encoding a glycosyltransferase under control of a tumor specific or tissue specific transcriptional control element. The glycosyltransferase typically forms a determinant on the cell surface to which some or all humans have naturally occurring antibody. In this manner, cancer cells will be culled on an ongoing basis by antibody already present in the circulation, without the need to follow the vector with an effector agent.
One embodiment of the invention is a polynucleotide as already described. Suitable glycosyltransferase enzymes include but are not limited to histo blood group A or B transferase from any upper primate (particularly human), and xcex1(1,3)galactosyltransferase (xcex11,3GT) of any mammal that forms the Galxcex1(1,3)Gal xenoantigen.
The transcriptional control element can be a tissue specific promoter, as exemplified below. Alternatively, the control element can be a tumor specific promoter, as exemplified below. Of particular interest is the promoter for telomerase reverse transcriptase (SEQ. ID NO:1). The polynucleotide can take the form of a viral vector (for example, adenovirus, herpes virus, or retrovirus), naked DNA, or a lipid composition (for example, a neutral or anionic lipid envelope, or a cationic liposome or micelle) that has a DNA or RNA component.
Polynucleotides of the invention can be used to prepare a medicament for human treatment, especially for conditions associated with hyperproliferation, such as cancer and other neoplasias.
Another embodiment of the invention is a polypeptide with glycosyltransferase activity, which comprises a consensus of mammalian xcex11,3GT sequences, or a humanized xcex11,3GT sequence, or catalytic subfragment thereof.
Also provided is a method of killing a cancer cell, comprising combining the cancer cell with a polynucleotide as already described. The invention includes a system for testing and manufacturing the glycosyltransferase vectors of this invention. The invention can be used for treating cancer in a subject by administering to the subject a polynucleotide as already described.
Other embodiments of the invention will be apparent from the description that follows.